MOLECULAR-CLONING AND FUNCTIONAL-CHARACTERIZATION OF RAT PLASMA-PROTEIN-S

Plasma protein S is a cofactor of activated protein C (APC) in the reg ulation of the blood coagulation system. Rat protein S homogeneously p urified from plasma showed cofactor activity for rat APC, but not for human APC when the APC cofactor activity was assayed using protein S- and C4b-binding protein (C4BP)-depleted human plasma. Rat plasma prote in S was separated by gel chromatography into two forms, a free form a nd a form complexed with C4BP. Rat protein S forms complexes with rat and human C4BP in a solid-phase model with apparent dissociation const ants (K(d)s) of 6.7 X 10(-8) and 1.2 X 10(-8) M, respectively, in the presence of 5 mM Ca2+. Human protein S also forms a complex with solid -phase human and rat C4BP with K(d)s of 6.3 X 10(-9) and 2.7 X 10(-8) M, respectively. Human C4BP strongly inhibited the APC cofactor activi ty of both human and rat protein S, whereas rat C4BP was only weakly i nhibitory. The degree of the inhibitory activity of C4BP appears to de pend on the affinity between protein S and C4BP. In order to evaluate the structure-function relationship of the rat protein S, the complete cDNA sequence of rat protein S was determined. This cDNA of 3,315 bp was composed of a 103-bp 5'-noncoding region, a 2,028-bp coding region that encodes a preprosequence of 41 amino acids, a mature protein S o f 634 amino acids and a stop codon, and a 1,184-bp 3'-noncoding region . The rat mature protein S consisted of domains with distinct function s similar to those of human protein S, and with two potential Asn-link ed glycosylation sites. The amino acid sequence of the mature form of rat protein S showed 80.4, 78.7, and 79.7% identity with those of huma n, bovine, and rabbit mature protein S, respectively. These findings s uggest that despite the species-specificity of the APC cofactor activi ty of rat protein S, it is structurally very similar to human protein S. Expression of rat protein S mRNA (approximately 3.5 kb) was demonst rated by RNA blot analysis not only in the liver, but also in the lung , spleen, testis, and uterus of rats.